This invention relates generally to a process for the preparation of peptides and peptide amides which contain sulfated tyrosine and more specifically to the solid phase synthesis of cholecystokinin (26-33), herein referred to as CCK-8, which has the following structure: EQU L-Asp-L-Tyr(SO.sub.3 H)- L-Met-Gly-L-Trp-L-Met-L-Asp-L-Phe-NH.sub.2 I
Ondetti and Pluscec (J. Am. Chem. Soc., 92, 195, (1970); J. Med. Chem., 13, 349 (1970); see also: U.S. Pat. Nos. 3,723,406; 3,778,429; 3,835,315; and 3,892,726) synthesized CCK-8 and a series of analogs by the methods of solution phase peptide chemistry, and found that CCK-8 as well as some analogs have a considerably higher potency than the parent molecule, CCK-33. When administered in doses of ca. 10.sup.-6 mg/kg, CCK-8 is a useful diagnostic agent for the examination of the contraction of the gall bladder and of pancreatic secretion (cf. U.S. Pat. No. 3,892,726). Investigations have also revealed that CCK-8 exerts a strong releasing action on the muscle sphincter Oddii; and thus, can be used with good results to alleviate the spasms occurring after gall bladder operations (Ondetti, Rubin, and Engel, J. Am Digestive Diseases, 15, 149 1970). More recent investigations have shown that CCK-8 exerts a strong anorectic effect (Crawley, Rojas-Ramirez, and Mendelson, Peptides, 3, 535 (1982) and references therein).
Ondetti and Pluscec (see references above) described a process for the preparation of CCK-8 from the protected octapeptide amide which was obtained from intermediate compounds prepared by the known methods of solution phase peptide chemistry. Overall yields reported for CCK-8 by these authors were in the range of 1-2% based on the starting phenylalanine amide. By a somewhat modified procedure, Penke, et al., (U.S. Pat. No. 4,102,878) described a process whereby CCK-8 was produced in 6.5% overall yield from phenylalanine amide by the known methods of solution phase peptide chemistry through an improved sulfation procedure. Sipos, U.S. Pat. No. 3,714,140 discloses the solid phase synthesis of peptides and gives as an example the synthesis of CCK-8. However, it is believed that CCK-8 cannot be synthesized by the Sipos method. For example, the cleavage reagent employed (Example 1B, HBr in trifluoroacetic acid/dichloromethane) generates C-terminal peptide acids not C-terminal peptide amides as is CCK-8. In addition, no mention was made as to how the peptide was sulfated or when. A method for the solid phase synthesis of the C-terminal fragments Asp-Phe-NH.sub.2 and Trp-Met-Asp-Phe-NH.sub.2 of CCK-8 has appeared (Gaudreau, et al., In "Peptides: Synthesis - Structure - Function" (Rich and Gross, eds.), pp. 193-195, Pierce Chemical Company, Rockford, Ill., 1981). Although the Gaudreau, et al., method does not describe the synthesis of CCK-8, it does point out some of the inherent problems associated with a synthesis of CCK-8 by the solid phase method. For example, when Boc-Trp(For)-Met-Asp(OPa)-Phe-OCH.sub.2 -Resin, where Boc is tert-butyloxycarbonyl, For is formyl, and OPa is phenacyl ester, was treated successively with 0.1M hydrochloric acid in formic acid for Boc removal, 1M sodium thiophenoxide in DMF for OPa removal and 30% ammonia in methanol for For removal and ammonolysis of the peptide from the resin, the major product isolated (60%) was not the desired product but Trp-Met-Asp(Phe-NH.sub.2)-NH.sub.2 is which PHe-NH.sub.2 had been transferred to the beta-carboxyl of Asp while the alpha-carboxyl of Asp had been amidated.
The inventors, in an earlier attempt to develop a solid phase synthesis of unsulfated CCK-8, synthesized the peptide resin Boc-Asp(OtBu)-Tyr-Met-Gly-Trp(For)-Met-Asp(OCH.sub.2 -PAM-Resin)-Phe-NH.sub.2 where OtBu is tert-butyloxy and OCH.sub.2 -PAM is 4-(oxymethyl)phenylacetamidomethyl. The peptide was attached to the resin through the beta-carboxyl of Asp. Treatment of the peptide resin with 50% trifluoroacetic acid in dichloromethane for Boc and OtBu removal and then with 10 equivalents of sodium hydroxide in 70% isopropanol for For removal and cleavage of the peptide from the resin resulted in the generation of Asp-Tyr-Met-Gly-Trp-Met-Asp(Phe-NH.sub.2)-OH while none of the desired product, Asp-Tyr-Met-Gly-Trp-Met-Asp-Phe-NH.sub.2, was detected.
Accordingly, heretofore, no definitive solid phase peptide synthesis of CCK-8 has been described.
We have discovered a process for the synthesis of peptides and peptide amides which provides higher overall yields, for example, 29% CCK-8 after purification to analytical purity in a single chromatographic step.